Application note

QuickExtract – Rapid and efficient extractionof PCR-ready genomic DNA from plant andseed samples
Mike Freeman MD, Luke Linz PhD; LGC, Biosearch Technologies, Alexandria, USA

Introduction
The ability to rapidly screen large populations
is vital for breeding and characterisation oftransgenic plants. Genotyping by methods
based on BHQ™ Probes or KASP™ arewidespread but generally require nucleic acid
purification. A more cost-effective and lesstime-consuming approach is needed. The
QuickExtract™ Plant DNA Extraction Solution
provides a simple, rapid DNA extraction method
to prepare genomic DNA for high-throughput
processing. It is used for leaf or seed samples.
The extracted DNA is ready for amplificationbased analyses.
The extraction requires less than ten minutes
to prepare PCR-quality DNA with two simple
heating steps (see Figure 1). The procedure isconvenient and can easily be scaled to process
hundreds of samples in multi-well robotic
automation systems.

QuickExtract
Plant Solution
Add samples

Heat at 65 °C
for 8 minutes

PCR-readyDNAFigure 1. Overview of the QuickExtract workflow. After the two heating steps,
the released DNA can be used directly for amplification-based analysis, stored
at +4 °C for four weeks, or transferred to -20 °C for archival purposes.

This application note demonstrates thesuitability of using the QuickExtract Plant
DNA Extraction Solution for multiple plant
species. The requirement for grinding is also
investigated. Genotyping results are shown for
KASP and BHQ Probes. In addition, the effectof storage on the quality of the extracted DNA
was determined.

QuickExtract is part of the Epicentre™ product line, known
for its unique genomics kits, enzymes, and reagents which
offer high quality and reliable performance.

Application note

QuickExtract – Rapid and efficient extraction of PCR-readygenomic DNA from plant and seed samples
Materials, methods and resultsa) Determination of the impact of grinding
Whole seeds from wheat, tomato, pepper andhulled sunflower were ground or incubatedwhole with QuickExtract Plant DNA Extraction
Solution – 100 µL of QuickExtract Solution
was added to tomato and pepper seeds, 200µL was added to wheat seeds, and 300 µL
was used for sunflower seeds. The processed
samples were diluted 1:4 or 1:16 prior to PCRTomatoSunflower

Pepper

16x dilution

4x dilution

Wheat

amplification with KASP on the IntelliQube™.
Genotyping results of the four plant species
are shown in Figure 2. Grinding had little tono effect on cluster plot analysis for tomato
and sunflower seeds. However, grinding wasrequired for amplification of QuickExtracted
DNA from wheat seeds and inhibitory for PCR
with pepper seeds. The decision whether
to grind a seed sample or perform theextraction on whole seeds must be determined
empirically.

Ground Seed

Whole SeedNTC

Figure 2. The effect of grinding seed material. Wheat, Tomato, Sunflower and Pepper seeds were ground (red circles) or extracted whole (blue circles) with QuickExtract
Plant DNA Extraction Solution. Samples were diluted 4-fold (top) or 16-fold (bottom) prior to PCR amplification with KASP on the IntelliQube. Water was used as anegative control (NTC).

Application note

QuickExtract – Rapid and efficient extraction of PCR-readygenomic DNA from plant and seed samplesb) Evaluation of BHQ and KASP chemistries
for eight commercially important crop typesQuickExtract Plant DNA Extraction Solution
was added to samples of seed or leaf materialof eight crop types – Corn, Wheat, Rapeseed,Soy, Tomato, Pepper, Cotton, and Sunflower.

Crop

Extracts were incubated as in Figure 1,
diluted 2- to 8-fold, and PCR amplified on theIntelliQube with KASP and BHQ chemistries.
Table 1 lists the crops tested, sample type,grinding requirements, starting weight of thesample and volume of QuickExtract Plant DNA
Extraction Solution used.

Tissuetype

Processing

Grinding required

Seed

ChippedOptional

Leaf

Punches

Optional

Seed

WholeRequired

CornWheat

Rapeseed
Soy
TomatoPepper

CottonLeaf

Required

QuickExtract Buffer
added (µL)

~20 mg200~100 mg

400

1-2 punches

100

5 punches

200

20 punches

400~35 mg200~5-10 mg100

~20 mg200~45 mg

400

Seed

Whole

Optional (Grinding slighlybetter)

~5 mg

100

Leaf

Punches

Grinding not tested~5-10 mg

100

Seed

Half

Grinding not tested~80 mg

200

Seed

Whole

Optional

~3 mg

100

Leaf

Punches

Optional

5 punches200

15 punches

400

Seed

Whole

No Grinding

~8 mg

100

Leaf

Punches

No Grinding

5 punches200

15 punches

400

Whole

Required~100 mg

200

Seed
Leaf

Sunflower

Cut

Mass (mg)

Seed
Leaf

HulledRequired~50 mg200

Punches

Not tested

2 punches

200

Whole

Required~100 mg

200

HulledOptional~50 mg200-300

Punches

Not tested

2 punches

200

Table 1. Sample and pre-treatment of validated crops, seeds or leaves, grinding requirement conditions, starting weight and volume of required QuickExtract solution.
The guidelines show how much tissue to use, and whether grinding the sample must be determined empirically for each plant species and tissue type. For reference,
one punch is 6 mm in diameter. Additional optimisation may be required.

Application note

QuickExtract – Rapid and efficient extraction of PCR-readygenomic DNA from plant and seed samples
Rapeseed

CornPepper

WheatSunflower

Soy

Seed
Leaf

KASP

Leaf

BHQ

Seed

TomatoFigure 3. Cluster plots for KASP and BHQ chemistries. Results are shown using seven different crops – Tomato, Rapeseed, Corn, Pepper, Wheat, Sunflower, and Soy.
Analyses were performed using KASP and BHQ Probes.

c) Stability of extracted DNA for at least 4weeks at +4 ˚CWe assessed the stability of the extracted
DNA. Corn and tomato leaf samples wereprocessed with the QuickExtract Plant DNA
Extraction Solution. The DNA was PCRamplified using two different KASP assays foreach crop type. Amplifications were performed

Corn assay 2

Tomato assay 1

Tomato assay 2

1 month

OriginalCorn assay 1

immediately after sample processing. Sample
plates were stored at +4 °C. After one monthof storage at +4 °C the amplifications wererepeated. Figure 4 shows a comparison of thegenotyping data, using the original QuickExtract
Plant DNA Extraction Solution lysate, and thesame QuickExtract lysate one month later.
No significant difference in endpoint signal orcluster quality was observed.

Figure 4. Stability of DNA stored at 4 ˚C. DNA extracted from corn and tomato with QuickExtract was PCR amplified (original) with two KASP assays and then stored at 4 °C
for 1 month. The samples were then again PCR amplified against the same two assays (1 month).

Application note

QuickExtract – Rapid and efficient extraction of PCR-readygenomic DNA from plant and seed samplesConclusion
The QuickExtract Plant DNA Extraction Solutionallows for fast and simple genotyping of plant
samples. Tomato and sunflower seeds allow
genotyping without grinding. Wheat seeds
required grinding for amplification, whereas
grinding of pepper seeds was inhibitory for
PCR. Leaf material for these crops showedsimilar results for the seed material (data notshown). We recommend that the necessity
or requirement for grinding be determinedfor each sample type. Also, performing serial
dilutions of the QuickExtract extract after
sample processing is advised, for example 1:4,1:8, 1:16, to determine the optimal amount ofprocessed sample volume for your downstream
application. Finally, it is important to emphasisethat seeds must be cleaned efficiently ifgenotyping, since interpretation of results may
be complicated by any non-plant material
adhering to the seed coat.
Results show that the QuickExtract Plant DNA
Extraction Solution may be used to extract
DNA from leaf or seed material from multiple
types of plants for endpoint PCR applications.
Good genotyping results and clusters, using

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GEN/0581/MW/0619

For Research Use Only. Not for use in diagnostic procedures.

FM 598408

KASP and BHQ Probes for different plantspecies, were generated. Parameters to beconsidered for each crop and sample (leaf orseed) are: initial sample mass, whether grindingis necessary, volume of the QuickExtract Plant
DNA Extraction Solution required and dilution ofthe QuickExtracted sample to use forthe assay.
In addition, it was shown that very good
genotyping results can be achieved even after
storing the original lysate four weeks at +4 °C.
This application note shows that QuickExtract
Plant DNA Extraction Solution provides afast and simple method to prepare genomic
DNA for KASP genotyping, or use of BHQ
Probes – all in a single tube, without the useof toxic chemicals, columns or precipitation
and resuspension steps. The method is
ideally suited to high-throughput applications
employing a liquid handler combined with theuse of a programmable heating block orwater bath.